Pitch degradation with white rot fungi

ABSTRACT

The fungi Schizophyllum commune, Trichaptum biforme and Phanerochaete gigantea are useful in reducing the pitch content of pulps and pulpwoods used in making cellulosic products.

This is a continuation of application Ser. No. 08/034,443, filed Mar.19, 1993 now abandoned.

The present invention relates to the use of certain fungi in thereduction of the pitch content of materials used in the manufacture ofcellulosic products.

Wood is a complex material composed of cellulose, hemicellulose, ligninand wood extractives or a resinous material commonly called "pitch","resin" or "wood resin". The composition of pitch has been studied andis reported widely in the literature, e.g., Wood Extractives and TheirSignificance to the Pulp and Paper Industry, Chapter 10 "Wood Resins" byD. B. Mutton; W. E. Hillis, Ed, Academic Press, N.Y. (1962).

In the production of products from wood pulps, the presence of pitch isundesirable as due to its viscosity and tenacity it frequently formsdeposits which are difficult to remove, causing relatively frequent andlengthy periods of down-time for cleaning, as resins tend to accumulateas deposits on strainer plates, filters, and throughout paper processingapparatus. It is well-known that pitch may also discolor pulp and paperformed therefrom if allowed to accumulate too long before cleaning.Other drawbacks are known in the art, e.g. waste stream pollution.

In Nilsson, et al., U.S. Pat. No. 3,486,969, it is disclosed thatcertain fungi may be used to inoculate wood chips to reduce the resincontent therein and the pulp therefrom while minimizing degradation ofthe other components of the wood, especially cellulose andhemicellulose. The species of fungi therein disclosed however, areapparently all mold type or surface forming fungi which, whendiscoloring the wood, produce essentially a surface or superficial stainwhich may be readily planed off (see J. S. Boyce, Forest Pathology, 3rd.Ed., 1961, McGraw-Hill Book Co. at pp. 493-512, especially 496-497).Such fungi have failed to achieve practical success to our knowledge.

In published European patent application EP 03 87 187A2 (based on U.S.patent application Ser. No. 310,814, filed 13 Feb. 1989 now abandoned)there are described the application of certain wood-penetrating fungigenerally classed as Ascomycetes or Deuteromycetes to pulpwoods andpulps to reduce the pitch content thereof. Similarly usefulwood-penetrating fungal derivatives are also disclosed in publishedEuropean patent application EP 04 70 929 A2(based on U.S. patentapplications having Ser. No. 560,521, filed Jul. 31, 1990, now abandonedand Ser. No. 657,581, filed Feb. 19, 1991 now abandoned).

In copending U.S. patent application Ser. No. 889,796, filed Jun. 17,1992, there are described other strain derivatives of a preferredwood-penetrating fungus Ophiostoma piliferum which exhibit very goodpitch degrading and aggressive growth characteristics while growingwhite or colorless on treated substrates.

A succession of preferred and improved wood-penetrating strains of O.piliferum as above-described have demonstrated commercial capability andhave achieved commercial success. In addition to substantial savingsfrom pitch reduction, early indications of greater paper strength(translating into faster machine speeds) have been confirmed and thereare further indications of greater pulping efficiency, particularly forexample when used on substrates for chemical pulping, probably due tothe ability of the fungus to substantially open up resin ducts and rayparenchyma cells. The ability of such fungi to be useful practically isin part attributed to the ability of the fungi to grow competitively onnon-sterile substrates and not be excluded or dominated by other fungior organisms which naturally infect wood sources. In retrospect, one canat least theorize why the indicated wood-penetrating fungi are able tobe useful and provide the indicated advantages. For example, theindicated wood-penetrating fungi are known to be early colonizers ofdead wood and hence early contributors to the process of wood decay. Onemight therefore imagine that a major natural purpose of such fungi isthe substantial removal or reduction of resin in the wood, a processwhich would also open up the resin ducts and parenchyma cells to theinvasion of the later colonizing rotting fungi, such as the white rotsand brown rots which are, for example, commonly found in the fungalclassification Basidiomytes (Basidiomycotina). The ability of theindicated wood-penetrating fungi to dominate other fungi includingBasidiomytes when substantial resin is present perhaps ensures thattheir pitch-degrading purpose is served and would be consistent with thetheory that their primary natural purpose may be pitch degradation.

In the general field of research of the potential use of fungi andfungal enzymes in paper making, the Basidiomytes, particularly white rotfungi, have been of interest for their ability to degrade lignin andproduce lignin degrading enzymes. The original concept referred to as"biopulping" was founded on the idea of an early treatment of pulpwood,e.g. in the form of wood chips, to begin the process of pulping orlignin removal prior to entry into the pulp mill itself. A white rotfungi judged particularly suitable for such purpose is Ceriporiopsissubvermispora as described in U.S. Pat. No. 5,055,159. While the causeor mechanism of action of such fungus in obtaining its desirable effectsare indicated in the patent to be related to selective lignindegradation, we have noted that some reported benefits are alsosuggestive of those obtained by our above-indicated pitch degradingfungi. Consistent with our general understanding concerningBasidiomytes, the fungus Ceriporiopsis subvermispora does not grow wellon non-sterile substrates and the subject patent discloses thesterilization of the substrates prior to inoculation with the fungus.

An objective of the present invention is to expand the field of fungiuseful in degrading pitch in pulps and pulpwoods, and particularly innon-sterile substrates.

Another object is to provide pitch degrading fungi having or combiningdesired properties such as color effects, pitch-degrading ability, goodgrowth on non-sterile substrates, flexibility in temperature ofoperation, greater action or flexibility of action on different woodspecies and the like.

In accord with the present invention, it has been found that white rotfungi of the species Schizophyllum commune, Trichaptum biforme andPhanerochaete gigantea are desirably effective in reducing the pitchcontent of wood substrates, including particularly pitch innon-sterilized wood substrates.

Accordingly, the invention provides a method of reducing the pitchcontent of wood, particularly pulpwoods and pulps, said methodcomprising applying to pulpwood or pulp an inoculum of the fungusSchizophyllum commune or of the fungus Trichaptum biforme or of thefungus Phanerochaete gigantea, or a mixture of inoculums for such fungi,and thereafter maintaining the inoculated pulpwood or pulp underconditions allowing growth of the fungus for a time sufficient to reducethe pitch content of the pulpwood or pulp.

By the terms "resin" or "pitch"(which are used interchangeably) is meantthat complex mixture of hydrophobic substances in wood, commonly knownas pitch, which are soluble in neutral organic solvents, such asmethylene chloride, diethyl ether, benzyl alcohol and the like. Theseinclude the terpenes, the diterpene ("resin") acids, fatty acids andesters, glycerides and waxes as well as alcohols, hydrocarbons and othercompounds associated therewith. For purposes of this invention, thestandard Tappi extraction analysis using methylene chloride will sufficefor measuring the reduction in resins which is the object of theinvention.

Resin or pitch is a significant constituent of both softwood, such assouthern pine, conifers and cedars, and hardwoods, such as Betula andPopulus, and it may comprise as much as 4% weight percent or even moreof the feed sent to mechanical or chemical pulping processes, generally1.5 to 4.0% for most woods used for pulping. Softwoods generally containmore resin than hardwoods, with pines having among the highest resincontent among softwoods. In hardwoods, resin is located primarily in theray parenchyma cells which form much of the fiber fraction when wood ispulped. In softwoods, resin is contained in both the ray parenchymacells and also in resin ducts.

The invention may be generally applied to reduce the pitch content ofpulpwoods and pulps used in the manufacture of cellulosic products.

The term "pulpwood" as used herein means any harvested (cut down) formof a tree material used in making paper, cardboard or other cellulosicproducts such as viscose, but prior to pulping, and includes such formsas timber, logs, wood chips, sawdust and the like. The term "refinedpulpwood" means a pulpwood resulting from the application of mechanicaland/or shearing forces to whole pulpwood forms such as logs to obtain amultiplicity of high surface area, small pieces, such as wood chips andsawdust, which are introducible into a pulping process. The inventionmay also be applied to lignin-containing cellulosic materialsclassifiable as pulps which have yet to undergo sufficient treatment tosignificantly reduce its lignin content (and liberate contained pitch),in particular pulp which still retains 60% or more of its originallignin content, such as first stage mechanical pulp.

The invention may therefore be utilized in one aspect thereof to atleast partially reduce the resin component of refined pulpwood andincompletely refined pulps by applying to the pulpwood or pulp aninoculum of at least one of the indicated fungi, accumulating theinoculated pulpwood or pulp in a mass and maintaining the accumulatedmass under conditions which allow or promote fungal growth in the massfor a time sufficient to effect a reduction in the resin component ofthe pulpwood or pulp by the fungus. The invention may be applied tounrefined pulpwoods such as cut timber in debark or undebarked form byinoculating the timber, desirably at least partially scored in the caseof undebarked timber, and maintaining the timber for a time sufficientto allow growth of the fungus on and into the wood substrate and effecta reduction in the resin component thereof.

By the term "inoculum" and the like as used herein is meant any fungalmaterial which is sufficiently viable to result in growth of the funguswhen applied to the substrate. Typical fungal inoculums include fungalcultures or preparation obtained from a fungal culture, desirably from abiologically pure culture. The basic structural unit of most fungi inthe fungal filament or "hypha". In aggregate, these filaments comprise afungal body call "mycelium". Fungi typically reproduce asexually bymeans of spores called conidia which are given off by the mycelia, ormay reproduce sexually by means of basidiospores or chlamydiospores. Allsuch forms and fungal elements, e.g. mycelia and spores, may be suitablyused as inoculum in the invention. An inoculum form may be provided byculturing the fungus in any of several conventional ways. Solid orliquid culturing media may be used as desired or required, preferablyliquid media. Culturing of the fungus under conditions favoring sporeformation is usually preferred when possible, and the generallypreferred inoculum will contain a large number of spores resulting fromthe fungal culture.

The inoculum may be in solid or liquid form. Whole liquid cultures orportions thereof may be used, e.g. mixtures of mycelia and spores. Whena high content of spores is available in the culture, the product may belyophilized (freeze-dried) to obtain a dry inoculum in which sporesconstitute the viable component to generate the fungus afterinoculation. Inocula in the form of concentrates to be diluted withwater for application are generally stored at temperatures which willpreserve desired viability. Liquid forms are usually stored frozen,typically at temperatures of from -5° C. to -80° C., more usually -10°C. to -75° C. Dry forms are similarly stored although lyophilized formscontaining spores as the operable inoculum are often more stable and maybe stored at higher temperatures than counterpart liquid forms. Inoculumcompositions may comprise other ingredients such as preservatives andstabilizing agents or inert carriers introduced in certain types ofdrying processes.

The inoculum may be applied to the wood substrate in a variety ofmanners. Typically, the inoculum is applied in a systematic ormethodical manner. For example, the inoculum is distributed at intervalsin the mass of refined pulpwood, or on the outer surface of a cuttimber, preferably at regular intervals. More preferably, the inoculumis distributed in a homogeneous or uniform manner, i.e. substantiallythroughout the mass of refined pulpwood. However, it is not necessarythat each individual wood chip, sawdust particle and the like beinoculated. As little as 10% or even less but preferably about at least20%, more preferably at least about 50%, of the individual pieces can beinoculated since the uninoculated pieces are accumulated in contact withthe inoculated pieces. Upon growth, the infection will spread veryeasily.

A thorough or uniform inoculation of a mass of wood chips is generallyreflected by the fact that the fungus grows substantially throughout themass. However, it may happen that some part of the mass, particularlythe outer layer of a pile of refined wood pulp, will show little growthcompared to the rest of the mass, or no growth at all, although it hasbeen inoculated.

In one preferred embodiment, the inoculum is sprayed onto wood chips orsawdust as they are discharged from the refining operation but beforebeing accumulated into piles. For example, a wood chipping apparatus isgenerally provided with conveyor means which receive the newly preparedchips and convey them to the accumulating pile. A spray applicatorcontaining the inoculum preparation may be conveniently adapted to theconveyor, preferably at the junction with the chipper when the chips areairborne e.g. free falling or tumbling, or at the very end of theconveyor so that chips are sprayed just before falling from theconveyor.

Alternatively, the inoculum may be applied to the wood chip pile in thecourse of its accumulation by more or less continuous spraying over theaccumulating pile.

When treating pulps or refined pulpwood, the dosage applied may varydepending upon several factors such as the wood being treated, conditionor age of the wood, growth conditions, desired treatment time and thelike. In general, satisfactory results can be obtained upon applicationof an inoculation containing from 0.5 to 10 grams of mycelia (wet weightof dewatered mycelia, see Example 1) per 100 grams of pulp or pulpwood,preferably from 1 to 5 grams of mycelia per 100 grams of substrate to betreated. Such mycelia prior to dewatering may be prepared as describedin Example 1 or Example A, below, preferably Example A, and may containspores. Dosage of an inoculation based predominantly or solely on sporesmay be routinely determined and can be indicated to range from 10⁶ to10¹⁰ CFU (colony forming units) per kilogram of substrate, more usuallyfrom 10⁷ to 10⁹ CFU per Kg. The inoculum dosage will generally beapplied in a water-diluted sprayable composition, for example, acomposition to be applied in a volume of from 20 to 60 ml. per Kg. ofsubstrate. The fungus is preferably applied to freshly cut or refinedpulpwood or freshly cut substrates frozen or stored at reducedtemperatures until treatment, or the substrate sterilized. When appliedto non-sterile pulpwood which has been allowed to age before treatment,e.g. wood chips which were produced about 5 days or more beforetreatment, it may be desirable to increase the inoculum dosage to thehigher end of the dosage range in order to avoid or suppress thebackground growth of fungi which naturally infected the wood prior toinoculation.

In another embodiment, chips which have been previously inoculated andincubated according to the invention may be dispersed into fresh chipsto effect or enhance inoculation. Such an inoculum is likely to be notbiologically pure. However, it reflects the previous inoculation as atleast 40%, preferably at least 50% of the inoculum is the desiredfungus.

After inoculation, the accumulated mass is maintained under conditionswhich will allow or promote the growth of the fungus substantiallythroughout the mass. Given the fact that the invention will in mostcases be likely to be practiced in open air and the mass thereforesubjected to a wide variety of weather conditions, the maintenance ofany given set of ideal conditions throughout the entire treatment periodis usually too difficult to achieve and is often unnecessary inpractice. It is generally sufficient that the mass be substantiallymaintained at a temperature at which the fungus grows while avoidinghigher temperatures at which the fungus dies. While our fungi mayexhibit some reasonable growth at or below 0° C. it will generally bemore suitable to have a temperature of at least 10° C. such as atemperature of from 10° C. to 40° C. more preferably of from 15° C. to33° C., most preferably of from 22° C. to 28° C.

In mild or warm weather conditions, it is not necessary to influence theenvironmental temperature and the inoculated mass may be left to standin open air without special maintenance. In cold weather conditions, itmay be desirable to provide the inoculated mass with means formaintaining the more suitable temperatures. This may be a heat-retainingcovering placed over or on the inoculated mass such as a large plasticsheet. Alternatively, the ground base on which is placed the inoculatedmass may be provided with heating pipes or a plurality of openings forreleasing warm air or steam. In a similar manner, a concrete "igloo" orsimilar structure which can be internally heated and emit radiant heatmay be used to support the accumulated mass of pulpwood. When providingheating means, it would also be desirable to control the moistureconditions to avoid an excessive dryness. In view of this, means forventing the heat or steam would be adequate. However, due to the heatgenerated in an accumulated mass from fungal growth and other microbialor natural effects, operation under many cold weather conditions mayproceed satisfactorily with little or no assistance.

The period of time during which the inoculated refined pulpwood mass istreated may vary considerably depending upon a number of factorsincluding the desired extent of resin removal, the temperature andmoisture conditions, the extent of inoculation and the like. However,satisfactory results may generally be obtained after a period of timeextending from 3 to 40 days, preferably from 4 to 30 days. Underpreferred conditions, very effective results e.g. a pitch reduction ofabout 20% or more, may be obtained 4 to 20 days after the inoculation,more usually 5 to 15 days.

Treatment of unrefined pulpwood, such as cut timbers, will usually besomewhat longer than that of refined pulpwood and may extend up to 2months. However, treatment of pulps and pulpwoods with the indicatedfungi generally should be conducted for periods which effect desiredpitch reduction while avoiding excessive periods which might result inany substantial attack on the cellulose component of the substrate(s).Dosages for unrefined pulpwood may be similar to those for refinedpulpwood and applied over from 10% to 100% of available surfaces, moreusually over 15% to 50% of the available surfaces.

The fungi used in carrying out the invention are previously knownspecies and may be obtained in a known manner, e.g. by isolation fromwood sources on which they grow in nature. While some variation amongstrains can be expected depending on factors such as the wood sourcefrom which they may be isolated, our fungi demonstrated remarkablegrowth on both unsterilized Southern Yellow Pine and Aspen and can beexpected to grow well on other wood types commonly used in makingcellulosic products. Naturally occurring isolates of our fungi can bemodified by various known means of strain selection, mating and mutationwithout losing their identifying species characteristics. Hence, ourpreferred natural isolates have been deposited with the NorthernRegional Research Center (NRRL), as detailed below, but it will beapparent that the same can be modified and that preferred fungi willinclude not only such isolates but also all other isolates andmodifications which substantially possess at least the pitch degradingand growth properties on unsterilized Southern Yellow Pine and Aspenthat are possessed by the deposited strain. The fungi used in theinvention will grow white or essentially colorless on pulpwood and pulp.Since they may be used to largely or completely dominate other darkergrowing fungi which naturally infect unsterilized substrates, the fungiof the invention may be used to produce a product requiring lessbleaching to obtain the final paper product.

DEPOSITS

We have under the Budapest Treaty deposited with the Northern RegionalResearch Center (NRRL) at Peoria, Ill., U.S.A. the following fungirefined to herein, which deposits were assigned the Accession Numbersgiven below along with their date of deposit.

    ______________________________________                                        Fungi          Accession No.                                                                              Deposit Date                                      ______________________________________                                        Schizophyllum commune                                                                        NRRL 21056   March 16, 1993                                    Trichaptum biforme                                                                           NRRL 21055   March 16, 1993                                    Phanerochaete gigantea                                                                       NRRL 21054   March 16, 1993                                    ______________________________________                                    

The above deposited fungi were all obtained as natural isolates fromfallen timber in the State of Minnesota, U.S.A., but all can be obtainedfrom a variety of other global locations. The S. commune and T. biformewere isolated from a hardwood and the P. gigantea was isolated from ared pine. It is noted that Trichaptum biforme has in the past also beenreferred to as Polyporus pergamenus and Hirschioporus pargamenus, seeGilbertson et al., North American Polypores, Vol. 2, Fungiflore, Oslo,Norway 1987, pages 770-772 and Otjen et al., "Selective Delignificationof Birch Wood (Betula papyrifera) by Hirschioporus pargamenus in theField and Laboratory", Holzforschung 40(1986), 183-189. Also,Phanerochaete gigantea has also been known in the past as Peniophoragigantea, see Burdsall, H. H., Jr., "A Contribution to the Taxonomy ofthe Genus Phanerochaete", Mycological Memoir, No. 10, J. Cramerpublishers, Braunschweig, Germany (1985).

EXPERIMENTAL

General Procedures: Cultures and Inoculation:

various evaluations are made on pulpwood substrates to determine pitchreduction and growth. For evaluation of softwood characteristics,sterile and non-sterile Southern Yellow Pine wood chips were used. Forevaluation of hardwood characteristics, sterile and non-sterile aspenwood chips were used. Wood chips are stored at 5° C. prior toevaluation. Each evaluation was performed on substrates of the same woodspecies and upon wood chips samples which were obtained from the samewood chip source. For each test, individual sample lots of wood chipswere first weighed, after which the wood chip samples to be sterilizedwere heated in an autoclave at 121° C. for about 20 minutes and allowedto cool to room temperature prior to the initiation of a test. The woodchip samples which were to be in non-sterile form were untreated andused in their natural condition. Individual sample lots were prepared byplacing measured amounts of wood chips into individual transparentplastic bags; the bags were of sufficient size such that they werecloseable (although not hermetically sealable). The use of a transparentbag allowed for the visual inspection of the growth of chips, and tofurther allow for admission of ambient light to the sample of wood chipsbeing evaluated.

A YNPD liquid culture medium was prepared using the followingconstituents (amounts are grams per liter of liquid culture mediumproduced):

    ______________________________________                                        10 g               glucose                                                    10 g               malt extract                                               2 g                peptone                                                    2 g                yeast extract                                              2 g                KH.sub.2 PO.sub.4                                          1 g                asparagine                                                 1 g                MgSO.sub.4.7H.sub.2 O                                      ______________________________________                                    

which are added in sequential order to one liter of deionized distilledwater, and subsequently autoclaved at 121° C. for about 20 minutes, andallowed to cool to room temperature. Afterwards, 1 mg. of thiamine isadded to the other constituents, after which the YNPD media was readyfor use.

Using the YNPD culture media prepared as indicated above, each of thefungi was prepared under the following general conditions:

(a) samples of the particular fungus were used to inoculate sterilepetri dishes which contained the YNPD culture media as prepared above,and the dishes were covered;

(b) the inoculated YNPD culture media was maintained at room temperature(approximately 20° C.) until it was visually discernible that theinoculated fungus had grown well upon the YNPD culture media in the formof mycelial mats (about 5 days);

(c) after good growth had been observed, the mycelial mats were thenremoved in hand (covered with a rubber glove) from the petri dish, themat squeezed in hand until essentially no further water was emitted andthe squeezed mat weighed to determine the "wet weight". The squeezed ordewatered mat was introduced into a clean laboratory beaker where it wasthen homogenized with the addition of between 5-10 ml. of distilledwater to form a pipetteable slurry which could then be removed from thebeaker and used to inoculate a substrate; and

(d) the contents of the beaker were then introduced into a graduatedcylinder to determine the volume of the pipetteable slurry, and oncedetermined, the contents were returned to the laboratory beaker, fromwhence they were withdrawn for inoculation of samples.

The inoculation of a sample of wood chips was done by injecting thecontents of the pipette containing 2-5 grams wet weight of the mycelialmat for each 100 grams of wood chips, after which the open end of thebag was folded over, and the contents of the bag shaken and tumbled soto maximize the number of chips that came into contact with theinoculant. The folded over end of the bag was stapled at two places. Allinoculated wood chip samples were then placed on a laboratory benchtopat room temperature for the periods indicated in each specific test.Each test was performed on two to five samples; reports of the growth offungi reported herein are the average of these plural results.

Pitch Content Evaluations:

Evaluation of the pitch content of substrates was determined accordingto standard TAPPI Procedure T204 OS-76 which provides resultsexpressible as milligrams of pitch content per gram of substrateextracted with "DCM" which is methylene chloride. In accordance with theTAPPI Procedure, as used on a substrate such as wood chips, the treatedchips are splintered with pruning shears to a width of about 1 cm., thendried overnight at 60° C. and then ground into sawdust using aThomas-Wiley Intermediate Mill with 10-mesh screen (10 gauge wirescreen), and then dried again overnight at 60° C. Three grams of thedried sawdust are combined with 30 ml. of DCM and the resulting mixtureis agitated overnight (about 15 hours) at room temperature(approximately 20° C.). The liquid medium is pipetted from the mixture,filtered through an organic filter having a pore size of 0.45 μm, andthen the liquid is allowed to evaporate at room temperature overnight ina tared (preweighed) dish. The dish residue is then heated in anair-circulation oven at 60° C. for 30 minutes to further remove anyresidual DCM, after which the dish is allowed to cool to roomtemperature and reweighed; the weight of the remaining residue, viz.,the remaining pitch, is determined and expressed in units of milligrams(mg.) and correlated to the amount of the original sample beingevaluated so to provide an expression of mg. of pitch per gram ofsubstrate wood chip, or in the alternative as the percent DCMextractables present in the substrate wood chip sample, which result isequated to and taken as the percent of pitch in the substrate (%extractives). Pitch evaluations may be conducted on both sterile andnon-sterile substrates. Evaluations on sterilized substrates willusually eliminate any possible influence of other organisms whichnaturally infect the substrate. An evaluation on a sterilized substratecan be generally considered the more objective measure of the fungus toreduce pitch on a particular substrate. However, whether conducted on asterilized or non-sterilized substrate, pitch reduction is generallyevaluated relative to an untreated control which is sterilized (forsterilized or substrate tests) held in the frozen state during the testperiod (non-sterilized substrate evaluation). In general, it is desiredto achieve a pitch reduction relative to such a control of at least 20%in no more than 21 days after inoculation, preferably in no more than 14days. Particularly good results are indicated when pitch is reduced 25%in no more than 21 days, and especially when such reduction is achievedin no more than 14 days.

Growth Evaluations:

Evaluations of the growth of the fungus is made as uniformly as possibleand in a manner as nearly identical as possible for all of theindividual samples being evaluated for each of the several tests wherethe growth is to be determined. Evaluation is done using simple visualobservation with a protocol applied on a consistent basis and carriedout at each evaluation interval (where an intermediate evaluation isperformed during a test) and at the end of each test. The protocol isbased on color categories of possible fungal growth which can beobserved or ascertained on each individual wood chip or substrate withthe unaided eye at normal reading distance. When the substrate issterilized, only one color category, that of the invention candidate,will be recognized and the protocol involves simple visual inspection ofall wood chips to determine the number or percentage of chips which showvisible growth of candidate fungus. When the growth evaluation iscarried out on non-sterile substrates, different color categories willbe usually recognized to distinguish between the invention or inoculatedfungus and those which naturally infested the substrate. The inoculatedcandidate, typically the lightest color, will be identified and thenumber or percentage of wood chips visibly exhibiting such growth willbe counted. Results reported below are given in terms of the percentageof the wood chips observed to exhibit growth of our desired fungus ineach test case. Treated, non-sterile wood chips may show growth in otherareas of the chips of other organisms, such as a black coloring fungi,and such background growth coloring may be separately recorded in asimilar fashion. Such background growth should not be taken as negatingotherwise positive growth results with the inoculated fungus, but themore desired fungal candidates are clearly those which best suppress ordominate over such background growth.

EXAMPLE 1

Growth on sterile and non-sterile Southern Yellow Pine:

An evaluation of fungal growth on Southern Yellow Pine was performed onboth sterile wood chip samples and non-sterile wood chip samples, thewood chips having been aged about 5 days. Each of the samples contained100 grams of wood chips, prepared as described above. An inoculant ofeach of the fungi was prepared as described above, and 5 grams ofmycelial mat (wet weight) were used to inoculate the 100 grams of chipsin the manner described above. The bags were then stored at roomtemperature for a period of 12 days. Evaluation of the growth of thefungi was performed at the second, fifth and twelfth day after theinoculation of the samples. The results of this growth on sterile andnon-sterile southern pine is reported in Tables 1 and 2 below. Withregard to the results on non-sterile substrates (Table 2) a minorbackground growth was observed on some wood chips after 12 days withsome of the background appearing under the otherwise white growing testfungus.

                  TABLE 1                                                         ______________________________________                                        Growth of fungi on sterile Southern Yellow Pine                                               2 days     5 days  12 days                                    Species         growth     growth  growth                                     ______________________________________                                        Schizophyllum commune                                                                         100%       100%    100%                                       Trichaptum biforme                                                                            100%       100%    100%                                       ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Growth of fungi on non-sterile Southern Yellow Pine                                           2 days     5 days  12 days                                    Species         growth     growth  growth                                     ______________________________________                                        Schizophyllum commune                                                                         100%       100%    90%                                        Trichaptum biforme                                                                            100%       100%    90%                                        ______________________________________                                    

EXAMPLE 2

Growth on sterile and non-sterile aspen:

Evaluations of the growth of the fungal species on aspen were performedon both aged sterile and aged non-sterile wood chip samples. Each of thesamples contained 100 grams of wood chips, prepared as described above.An inoculant of each of the fungi was prepared as described above, and 3grams of mycelial mat (wet weight) were used to inoculate the 100 gramsof chips. The bags were then stored at room temperature for a period of12 days. The growth evaluation was performed at the second, fifth, andtwelfth day after the inoculation of the samples. The results on sterileand non-sterile aspen are reported in Tables 3 and Tables 4, below. Withregard to the results on non-sterile substrates (Table 4), a minorbackground growth was observed on some wood chips after 12 days growth.

                  TABLE 3                                                         ______________________________________                                        Growth of fungi on sterile aspen                                                              2 days     5 days  12 days                                    Species         growth     growth  growth                                     ______________________________________                                        Schizophyllum commune                                                                          75%       100%    100%                                       Trichaptum biforme                                                                            100%       100%    100%                                       ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Growth of fungi on non-sterile aspen                                                          2 days     5 days  12 days                                    Species         growth     growth  growth                                     ______________________________________                                        Schizophyllum commune                                                                         75%        75%     100%                                       Trichaptum biforme                                                                            65%        75%      85%                                       ______________________________________                                    

EXAMPLE 3

Following essentially the procedure of Examples 1 and 2, the fungusPhanerochaete gigantea was evaluated for growth on both aged non-sterileSouthern Yellow Pine and aged non-sterile Aspen, except that theinoculum in each test was a low dose of 2 grams wet weight of myceliaper 500 grams of wood chips and the evaluation was carried out over 27days with an intermediate evaluation at 14 days. No background growthwas observed either at 14 days or at 27 days. The results are below inTable 5.

                  TABLE 5                                                         ______________________________________                                               Growth on Pine                                                                              Growth on Aspen                                          Fungus   14 Days   27 Days   14 Days 27 Days                                  ______________________________________                                        Phanerochaete                                                                          95%       100%      90%     100%                                     gigantea                                                                      ______________________________________                                    

EXAMPLE 4

Removal of Pitch in Hardwoods (Aspen):

The fungal strains were evaluated for their efficacy in the removal ofpitch in aspen and other characteristics. Control samples were alsoevaluated to provide a comparative indication. Control samples includeda non-inoculated control sample which was maintained frozen (-20° C.)throughout the period of the test, and a non-inoculated control samplewhich was maintained at room temperature. The ambient temperaturecontrol was used as an indicator of the effect on pitch reduction ofbackground organisms present on the non-sterile wood chip samples. Allevaluations were performed on 400 gram samples of non-sterile aspen woodchip samples after 14 days of growth after inoculation, with each testrun in triplicate and the results averaged (the wood chips had been agedabout 5 days prior to inoculation). For comparison, the tests alsoinvolved the fungal species Ophiostoma piliferum in the form of theproduct available under the registered trademark CARTAPIP® 97.

Each of the samples were evaluated for the amount of DCM extractable inaccordance with the protocol described TAPPI Procedure T204 OS-76.Analysis of the Klason lignin was performed upon selected aspen woodchip samples to provide an indicator of the degradation of lignin in thesample chips; quantitative determination of five principalmonosaccharides (glucan, mannan, arabinan, xylan and galactan) wasperformed on an absolute basis so to define the carbohydrate compositionof the wood. This Klason lignin analysis was performed generally inaccordance with the testing protocol of TAPPI T249 cm-85 "Carbohydratecomposition of extractive-free wood and good pulp by gas-liquidchromatography" (1984; TAPPI). In summary, Klason lignin analysisaccording to the TAPPI T249 cm-85 protocol is as follows; samples arehydrolyzed with sulfuric acid using a two-step technique; a portion ofthe hydrolyzate is then neutralized and the sugars present in the samplereduced with sodium borohydrate to the alditols, which are thenacetylated with acetic anhydride and pyridine, and the alditol acetatesthen dissolved in methylene chloride and then used for injection intothe gas chromatograph. Further, for selected aspen wood chip samples ananalysis of the carbohydrates was performed so to evaluate the extent ofcellulose and hemicellulose degradation.

Results of the samples being evaluated, % DCM extractives and % Klasonlignin are reported on Table 6, and the carbohydrate analysis ofselected samples are reported on Table 7, both below.

                  TABLE 6                                                         ______________________________________                                        Inoculant amounts, % DCM extractives and % Klason lignin                                 wet weight of                                                                 mycelia/400 g                                                                              % DCM      % Klason                                   Fungus     wood         extractives                                                                              lignin                                     ______________________________________                                        non-inoculated,                                                                          0            1.92       --                                         Frozen control                                                                non-inoculated,                                                                          0            1.61       18.0                                       ambient control                                                               Ophiostoma 4 × 10.sup.8 CFU.sup.a)                                                              1.37       --                                         piliferum                                                                     CARTAPIP.sup.R 97                                                             S. commune 21 g         1.33       17.9                                       T. biforme 22 g         1.29       18.1                                       P. gigantea                                                                              20 g         ca 1.30    --                                         ______________________________________                                         .sup.a) CFU is colony forming Units based solely on spore count for O.        piliferum (product only contains spores).                                

                  TABLE 7                                                         ______________________________________                                        Carbohydrate analysis                                                         Sample: arabinan xylan    mannan galactan                                                                             glucan                                ______________________________________                                        ambient 0.33     18.0     1.3    0.4    45.1                                  control                                                                       T. biforme                                                                            0.13     17.9     1.4    0.5    43.9                                  S. commune                                                                            0.23     18.4     1.2    0.5    45.3                                  ______________________________________                                    

As may be seen from the Klason lignin test results, fungal species ofthe invention were found not to appreciatively affect the lignin contentof the wood chip samples. Surprisingly, the fungal species of theinvention caused a significant reduction in the pitch content of thesamples, it being noted that CARTAPIP® 97 is regarded as a potentdegrader of pitch.

As may be seen from the results of Table 7, there was not an appreciableloss in the amount of carbohydrates in samples of aspen wood chips whichwere treated with our fungi as compared to the ambient control sample.Hence no reduction of cellulose and/or hemicellulose was indicated as aresult of the pitch reducing treatments.

EXAMPLE 5 GROWTH CHARACTER OF FUNGI IN LIQUID SHAKE FLASK CULTURE

Schizophyllum commune and Trichaptum biforme were each separately grownin shake flask liquid culture using 50 ml. of a malt extract/yeastextract medium prepared by dissolving 20 g. malt extract and 2 g. yeastextract in distilled water to a total volume of 1 liter. The medium wasinoculated with a small plug of mycelia from an actively growingmalt/yeast extract agar plate. The flask was shaken at 200 rpm at23°-25° C. for 5 days and a 1 ml sterile sample from each culture wasremoved for microscopic analysis. Both cultures showed a dense growth ofmycelial balls and the culture masses were also indicated to includefrom about 40 to 60% blastospores (about 40% for T. biforme and 50-60%for S. commune). Both products can be used as inoculum or processed invarious ways to produce inoculum forms, e.g. by homogenizing andfreezing for later use. Inoculum based essentially on the spore contentof the cultures may also be prepared by freeze drying.

What is claimed is:
 1. A process for reducing the pitch content ofpulpwood or of a pulp having at least 60% by weight of its originallignin content comprising applying to the pulpwood or pulp, one or moreinocula of one or more fungi capable of reducing pitch content andselected from the group consisting of Schizophyllum commune, Trichaptumbiforme, and Phanerochaete gigantea, the inoculation being of a numberand in an sufficient upon fungal growth from the inoculation to reducethe pitch content of the pulpwood or pulp, and maintaining theinoculated pulpwood or pulp under conditions which allow fungal growthfrom the inoculation for a time sufficient to effect a reduction of thepitch content of the pulpwood or pulp by the fungal growth.
 2. Theprocess of claim 1 in which the pulpwood unsterilized pulpwood.
 3. Theprocess of claim 2 in which the pulpwood in debarked or undebarkedtimber or logs.
 4. The process of claim 2 in which the unsterilizedpulpwood is unsterilized refined pulpwood, in which the inoculatedrefined pulpwood is accumulated in a mass and in which the accumulatedmass is maintained under conditions which allow fungal growth for a timesufficient to effect a reduction of the pitch content of the pulpwood bythe fungal growth.
 5. The process of claim 4 in which the fungus isSchizophyllum commune.
 6. The process of claim 4 in which the fungus ifTrichaptum biforme.
 7. The process of claim 4 in which the fungus isPhanerochaete gigantea.
 8. The process of claim 4 in which the refinedpulpwood is consists of wood chips and the inoculum is applied byspraying of wood chips with the inoculum prior to accumulation of thewood chips in the mass.
 9. The process of claim 8 in which the fungus isSchizophyllum commune having at least the ability to reduce pitch and togrow on unsterilized Southern Yellow Pine and Aspen that is possessed bythe Schizophyllum commune strain of NRRL Accession No.
 21056. 10. Theprocess of claim 8 in which the fungus is a Trichaptum biforme having atleast the ability to reduce pitch and to grow on unsterilized SouthernYellow Pine and Aspen that is possessed by the Trichaptum biforme strainof NRRL Accession No.
 21055. 11. The process of claim 8 in which thefungus is a Phanerochaete gigantea having at least the ability to reducepitch and to grow on unsterilized Southern Yellow Pine and Aspen that ispossessed by the Phanerochaete gigantea strain of NRRL Accession No.21054.
 12. The process of claim 8 in which the inoculated pulpwood ismaintained under fungal growth conditions for a period of from 4 to 20days after inoculation.
 13. The process of claim 1 in which the fungusis Schizophyllum commune.
 14. The process according to claim 13 whereinthe Schizophyllum commune is the strain of NRRL Accession No.
 21056. 15.The process of claim 1 in which the fungus is Trichaptum biforme. 16.The process according to claim 15 wherein the Trichaptum biforme is thestrain of NRRL Accession No.
 21055. 17. The process of claim 1 in whichthe fungus is Phanerochaete gigantea.
 18. The process according to claim17 wherein the Phanerochaete gigantea is the strain of NRRL AccessionNo.
 21054. 19. The process of claim 1 in which the inoculum is obtainedfrom a biologically pure fungal culture.
 20. The process of claim 1 inwhich the pulpwood is first stage mechanical pulp.
 21. The process ofclaim 1 in which the inoculated pulpwood or pulp is maintained underfungal growth conditions for a period of from 4 to 20 days afterinoculation.
 22. A biologically pure strain of a fungus of the speciesSchizophyllum commune with all of the identifying characteristics of thestrain of NRRL Accession No.
 21056. 23. A biologically pure strain of afungus of the species Trichaptum biforme with all of the identifyingcharacteristics of the strain of NRRL Accession No.
 21055. 24. Abiologically pure strain of a fungus of the species Phanerochaetegigantea with all of the identifying characteristics of the strain ofNRRL Accession No. 21054.